Assay of cyclic AMP-dependent protein kinase activity in canine myocardium: effect of coronary artery ligation on the cytosolic enzyme.

A procedure for the determination of the activation state of cyclic AMP-dependent protein kinase (cAMP-PK) in the cytosolic fraction of dog myocardium was derived from existing assays of this enzyme in tissues, a new element being the absence of added sodium chloride throughout the procedure. About 2/3 of the total cAMP-PK in normal dog ventricular myocardium was found to be present in the cytosolic (supernatant) fraction, the remaining 1/3 being particle-bound. The isoenzyme profile in the cytosolic fraction as determined by DEAE-cellulose chromatography revealed the presence of nearly equal amounts of the type I and type II isoenzymes. This distribution was also evident in experiments in which the RI and RII regulatory subunits were photoaffinity labeled with 8-azido- [32P] cAMP. Ligation of the left circumflex coronary artery in anesthetized open-chest dogs kept under artificial respiration caused increases in the cAMP-PK activity ratio (-cAMP/+cAMP) from a control value of 0.11 to 0.26 and 0.22, respectively, in the ischemic and in a nonischemic area of the left ventricle. This effect was clearly evident 2 min after ligation and was still observable in the ischemic area after 20 min. It was associated with increases in cAMP levels and in the activity ratio (-5' -AMP/+5' -AMP) of glycogen phosphorylase in the ventricular tissues. Pretreatment of the dogs with (+/-)-propranolol (2 mg/kg) abolished the cAMP and cAMP-PK responses, while the phosphorylase activity ratio remained elevated.